Confocal analysis of CD41 and VWF localization. (A) Fixed platelets from hTg^WT animals were allowed to adhere to glass coverslips and subjected to immunofluorescent staining. Maximum intensity projections of a deconvolved image stack were obtained using confocal microscopy. The raw confocal stack images were deconvolved with iterations limited to 10 using Huygens Essential Software. The presented image is a color merge of VWF (red) and CD41 (green) in hTg^WT platelets. VWF staining was obtained with a rabbit polyclonal anti–human VWF antibody and an anti–rabbit CY3 secondary antibody. The hTg^WT platelet surface was labeled using a rat anti–mouse CD41 antibody and an anti–rat Alexa488 secondary antibody. The circled platelet was further analyzed and is presented as a surface rendered image in Figure 2. (B) A higher magnification of the boxed region is shown for clarification. VWF labeling is restricted to platelet granules. (C) As described for panel A, wide-field microscopy of platelets from mice expressing a G233V GP Ibα subunit (hTg^G233V) is shown. (D) A higher magnification of an isolated region is shown for clarification.